RNA's modified nucleosides are essential to protein synthesis. However, too little is known of the biochemical and structural contributions of the 100 natural, post-transcriptional modifications to relate structure to function. Fundamental understandings of modified nucleoside functions have shown promise in identifying novel therapeutic targets in infectious disease, and in optimizing ribozyme, antisense and aptamer activities. Thus, this project's long-term objective is to understand how the biochemical and structural contributions of modified nucleosides impact protein recognition of RNA and its function in translation. tRNA, composed of physically and functionally separable domains that are easily investigated for modification-dependent structure/function relationships, is an excellent model for this study. We hypothesize that anticodon modified nucleosides provide a common architecture for stably recognizing mRNA coding triplets on the ribosome, while at the same time providing hydrophobic, hydrophilic and/or electrostatic properties as identity elements for aminoacyl-tRNA synthetase (aaRS) recognition. Using methods developed almost exclusively by us for the automated chemical synthesis of RNA with site- selectively placed modified and stable isotope labeled nucleosides, we have found three functionally important modified nucleosides that modulate anticodon loop architecture. To determine the structure/function relationships of other anticodon domain modifications, we will: 1) Identify and characterize the modified nucleoside-dependent ribosomal binding of tRNA anticodon domains that do not bind in their unmodified sequences; 2) Determine and characterize the properties of modified anticodon domains that are selectively recognized by peptides as mimics of aaRS; and 3) Characterize the structural properties contributed by modified nucleosides to the ribosomal binding and aaRS recognition of anticodons. Our approach will utilize a unique combination of chemical synthesis, biochemical assays of function and thermal denaturation, calorimetry, fluorescence, CD and NMR spectroscopy for determining stability and structure to distinguish those modifications required for ribosome binding and aminoacylation and their critical physiochemical contributions.